Utility of hTERT-driven overexpression of E2F-1 to induce selective apoptosis of human breast cancer cells

Abstract

3158 Background: The transcription factor E2F-1 normally induces cell cycle progression at the G1/S checkpoint, but when overexpressed will induce apoptosis. We have shown that CMV-driven overexpression of E2F-1 using an adenoviral vector (AdCMV-E2F-1) can induce apoptosis in multiple types of cancer cells independently of p53, p73 and caspases. We developed an adenoviral vector, AdhTERT-E2F-1, with E2F-1 under the control of the human telomerase reverse transcriptase (hTERT) promoter to minimize cytotoxicity to normal cells. We have shown that it induces apoptosis in colon cancer cells, and that its systemic delivery inhibits growth of liver metastasis in murine colon cancer models. Therefore, we wished to determine the ability of AdhTERT-E2F-1 to selectively induce apoptosis of breast cancer cells. Methods: Human breast cancer cells (MDA-MB-468) and immortalized breast epithelial cells (76NE6) were transfected (MOI of 2000) with AdhTERT-E2F-1, control vector AdhTERT-Luc, and mock transfected with PBS. Viable cells were counted at 24, 48, and 72 hours. At 72 hours apoptosis was measured by FACS, and protein was isolated from MDA-MB-468 transfected cells and subjected to a 10% SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were incubated with antibodies against E2F-1, poly ADP-ribose polymerase (PARP), Apoptosis Inducing Factor (AIF), caspase-8, and β-actin. Results: MDA-MB-468 cells transfected with AdhTERT-E2F-1 demonstrated 50% growth inhibition and a 4.8-fold increase in apoptosis as compared to controls (AdhTERT-Luc, PBS). However, transfection of 76NE6 cells with AdhTERT-E2F-1 did not cause growth inhibition or apoptosis as compared to controls. Western blot analysis of MDA-MB-468 transfected with AdhTERT-E2F-1 showed E2F-1 overexpression, PARP cleavage (hallmark of apoptosis), and upregulation of caspase-independent AIF and caspase-8. Conclusions: These findings demonstrate that AdhTERT-E2F-1 can cause selective apoptosis of human breast cancer cells. Thus, we believe that AdhTERT-E2F-1 has promise for development in the treatment of metastatic breast cancer. No significant financial relationships to disclose.